Human cytomegalovirus (HCMV) infection causes life-threatening disease in immunocompromised hosts and serious neurological impairment to developing fetuses in women infected during pregnancy. Proteins encoded by the HCMV major-immediate early (MIE) gene are essential to productive CMV infection and replication, but it is unknown how CMV commandeers host cell machinery to direct MIE protein synthesis. Our preliminary findings reveal that MIE gene expression is regulated at the splicing level, causing organ-specific MIE gene expression, protein synthesis and CMV infection in vivo, and novel interactions of CMV with host cell splicing apparatus. This proposed four-year approach is fundamentally different from previous MIE gene regulation studies that target the viral gene transcription, and is expected to be highly complementary with already-known information. Indeed, our long-term goal is to elucidate how CMV usurps host cell gene-splicing machinery to direct MIE gene synthesis, including: mechanisms that regulate splicing of CMV very early genes; the influence of MIE gene splicing on CMV replication in vitro and in vivo; the novel roles of nuclear domains (ND10 and SC) and their associated proteins in CMV gene regulation and viral replication, and how these influence CMV-induced neurodevelopmental pathogenesis. Our central hypothesis is that during early-stage infection, CMV counteracts host cell defenses including ND10 and its associated proteins, usurps the host cell splicing regulators PTB, U2AF and SC35, and activates early genes by interacting with specific cis-elements. The specific aims are: 1) To identify major cis-elements of the MIE gene that regulate MIE gene splicing, and determine the importance of splicing factors for HCMV replication. 2) Determine the biological importance of interactions between HCMV IE1/IE2 and cellular gene splicing regulators and of nuclear domains in regulating HCMV gene splicing. 3) Determine the influence of tissue- specific alternative splicing of the MIE gene upon organ-selective CMV pathogenicity in vivo, and the pathogenic influence of IE1/IE3 on embryonic neurogenesis in vitro and in vivo in mice. These studies will introduce novel concepts and findings that advance basic understanding of molecular virology, and will combine innovative in vitro and in vivo models and novel tools to elucidate key mechanisms of MIE gene splicing regulation that are critical to CMV gene expression and productive host infection. Positive impacts will include qualitative advances in understanding of molecular virology and CMV pathobiology in particular and also identification of new candidate targets for preventive and therapeutic intervention. Understanding the mechanisms CMV uses to regulate MIE gene splicing may thus drive development of new, selective anti-CMV therapeutic strategies, as the MIE gene products IE1 and IE2 are essential to CMV early/late gene expression and HCMV replication.